Pre-activation Based One-pot Synthesis of an α-(2,3)-Sialylated Core-Fucosylated Complex Type Bi-antennary N-Glycan Dodecasaccharide

Abstract

Synthesis of N-glycans is of high current interests due to their important biological properties. A highly efficient convergent strategy based on the pre-activation method for assembly of the complex type core fucosylated bi-antennary N-glycan dodecasaccharide has been developed. Retrosynthetically, this extremely challenging target is broken down to three modules: a sialyl disaccharide, a glucosamine building block and a hexasaccharide diol acceptor. The sialyl disaccharide was easily obtained by selective activation of a new 5-N-trichloroacetyl protected sialyl donor in the presence of a thiogalactoside acceptor. The hexasaccharide diol module was produced by double mannosylation of a fucosylated tetrasaccharide acceptor, which in turn was generated by glycosylation of a α-fucosylated disaccharide with a β-mannose containing disaccharide donor. The union of the three modules was performed in one pot giving the fully protected dodecasaccharide in high yield. This synthesis is characterized by minimum protective group and aglycon adjustment on oligosaccharide intermediates, thus greatly enhancing the overall synthetic efficiency. The modular feature of this strategy suggests that this method can be readily adapted to the synthesis of a wide variety of N-glycan structures.

Introduction

Glycosylation is one of the major types of postsynthetic modification of mammalian proteins, which include the attachment of oligosaccharides to asparagine (N-glycan) and to serine or threorine (O-glycan).[1] Unlike protein synthesis, the addition of carbohydrates to proteins is not under direct genetic control, which often leads to heterogeneous glycosylation patterns on the same protein backbone. The identities of carbohydrate moieties can have a profound effect on biological functions of glycoproteins such as their immunogenecities, stabilities, and affinities to receptors.[1–4] However, despite intensive studies, detailed structure-function relationships have not been established in many cases, mainly due to the difficulties in accessing sufficient quantities of these glycan structures.

We became interested in the synthesis of α-(2,3)-sialylated core-fucosylated complex type bi-antennary N-Glycan dodecasaccharide 1, which is one of the prototypical structures of mammalian N-glycans.[5] The dodecasaccharide 1 has been found on alpha fetoprotein (AFP) isolated from patients having hepatocellular carcinoma, a form of liver cancer. It is proposed that the structures of N-glycans can be a much more reliable marker to differentiate benign and malignant liver diseases.[6, 7] Similarly, studies on N-glycans linked to prostate specific antigen (PSA) suggest that α-(2,3) linked sialylated N-glycan on PSA can be potentially used to identify prostate cancer.[8] Dodecasaccharide 1 has also been found on the surface of erythropoietin.[9] The presence of fucose and sialic acid moieties in attached carbohydrates is believed to be important for the in vivo activity of erythropoietin.[10] There are intense interests in remodeling glycoproteins such as erythropoietin with homogeneous carbohydrate structures.[11] Thus, the ready availability of dodecasaccharide 1 can greatly facilitate the exploration of its glyco-biological functions and biomedical applications.

Although N-glycan structures can be assembled via several approaches,[12–23] total synthesis of dodecasaccharide 1 via chemical methods still presents a daunting task, which requires the construction of many difficult glycosyl linkages such as β-mannose, α-sialic acid, α-fucose as well as branching sequences. Moreover, the acid lability of the fucosyl linkage limits the scope of reagents that can be used. To date the only chemical total synthesis of the dodesaccharide in its fully protected form was accomplished by the Danishefsky group using a variety of glycosyl donors including glycals, thioglycosides, glycosyl sulfoxide, glycosyl fluoride and glycosyl phosphite. The tour-de-force total synthesis took 20 steps starting from protected monosaccharide building blocks with 13 steps and 3.1% overall yield for the longest linear sequence.[11, 24] Herein, we report our studies on an efficient chemical synthesis of dodecasaccharide 1 via the pre-activation based one pot method predominantly using p-tolyl thioglycosides to simplify building block design.

Results and Discussion

In order to enhance the overall efficiency, our synthetic sequence was designed to minimize protective group manipulations and aglycon adjustments on intermediate oligosaccharides while achieving high stereoselectivities and yields. Previously, we discovered that the reaction of a mannosyl diol acceptor with a thiomannosyl donor in the absence of a participating protective group on its 2-O position led to products as an anomeric mixture.[26] Therefore, the strategic disconnections between glycan rings C/D and C’/D’ in dodecasaccharide 2 were chosen, leading to ABC trisaccharide fragment 3 and the core DD’EFGH hexasaccharide 4 (Scheme 1).

Scheme 1.

Retrosynthetic analysis of dodecasaccharide 1.

For the installation of DD’ units in 4, to minimize the steric hindrance to the 3-O position of the E mannosyl unit, a popular strategy is to introduce the D’ unit first followed by protective group adjustment and glycosylation on the 6-O position.[12, 27] For higher efficiency, we decided to explore the possibility of double mannosylation[17, 28] of tetrasaccharide 6 by the 2-O acetylated thiomannoside 5. In tetrasaccharide 6, the formation of β-mannosyl linkage between ring E and F is crucial. β-Mannosides can be accessed via a variety of methods, including SN₂ inversion of β glucosides[15, 18, 29] intramolecular aglycon delivery,[27, 30, 31] insoluble silver salt promoted mannosyl halide reaction[32] and glycosylation by benzylidene bearing mannosyl donors.[33–35] The ground breaking work by Crich and coworkers has demonstrated high β selectivity can be obtained using benzylidene bearing mannosyl donors,[33–36] although the aglycon of the product typically needs to be further modified to convert it into a suitable donor for subsequent glycosylation to extend the chain.[20]

Our journey commenced from the preparation of the reducing end disaccharide 9 through fucosylation of diol acceptor 8[37] by donor 7 (Scheme 2a).[38] Although exclusive α selectivity was obtained when donor 7 reacted with acceptors containing secondary hydroxyl groups using N-iodosuccinimide/triflic acid or p-TolSCl/AgOTf promoter systems,[39–41] α:β mixtures were generated with acceptors bearing primary hydroxyl groups under these conditions. In order to enhance the α selectivity, we applied the in situ anomerization protocol (CuBr₂, tetrabutylammonium bromide)[27, 28, 42] first developed by Lemieux and coworkers.[43] Satisfactory yield (75 %) was obtained for the desired disaccharide 9, with its regio- and stereo-chemistry confirmed by NMR analysis.

Scheme 2.

The formation of the key β mannosyl linkage using the benzylidene bearing thiomannosyl donor 10 was examined next. Activation of thiomannosyl donor 10α in the absence of any acceptor (pre-activation)[44] by p-TolSOTf, formed in situ through the reaction of p-TolSCl and AgOTf,[44, 45] was followed by addition of thioglycosyl acceptor 11,[46] which formed disaccharide 12 in 57% yield (82% based on the amount of donor consumed) with an β:α ratio of 6:1 (Scheme 2b). The aglycon stereochemistry of the donor did not affect the reaction as donor 10β gave identical result as 10α. The newly formed glycosyl linkage in 12β was confirmed by the one bond coupling constant between the anomeric carbon and proton of the mannose moiety (¹J (C₁,H₁) = 157 Hz).[47] Disaccharide 12β was then directly used as a donor without any aglycon manipulations.

The reaction of disaccharide 12β and 9 promoted by p-TolSCl/AgOTf produced the desired tetrasaccharide 13 despite the presence of the bulky fucosyl group on the 6-O position of acceptor 9. However, treatment of 13 with PhBCl₂ and Et₃SiH for opening the benzylidene group[48] cleaved off the fucose unit. After several alternative reaction routes including postponing the introduction of fucose were explored, the most efficient way was determined to be adjusting the protective groups on disaccharide 12β. The p-methoxybenzyl (PMB) group in 12β can be oxidatively removed first followed by benzylidene opening.[19, 49] We discovered that mixing PhBCl₂ and Et₃SiH with 12β not only regioselectively converted the benzylidene group to 4-O benzyl but also removed the 3-O-PMB group simultaneously, leading to a diol which was subsequently protected as levulinoyl ester 14 (Scheme 2c, 77%). Reaction of the levulinoyl disaccharide donor 14 with fucosylated acceptor 9 proceeded smoothly in 86% yield. The two Lev moieties in the tetrasaccharide product 15 were selectively deprotected using hydrazine acetate giving diol 6 (Scheme 2c). Double mannosylation of 6 by donor 5 was explored next with our p-TolSCl/AgOTf promoter. A product with the same molecular weight as the desired hexasaccharide was isolated, which was found to be extremely labile to acid. NMR studies suggested that it was an orthoester. To overcome this problem, TMSOTf[50] was added at the end of the glycosylation reaction at 0 °C to rearrange the orthoester in situ. Gratifyingly, the desired hexasaccharide 16 was obtained in 77% yield following TMSOTf promoted rearrangement (Scheme 2d). Hexasaccharide 16 was then de-acetylated resulting in the core hexasaccharide diol acceptor 4.

Next we focused our attention on the assembly of the branching sequence. Sialylation is known to be notoriously difficult due to the low reactivity of the sialyl donor and the challenge in stereochemical control.[51] The presence of the carboxylic acid moiety in sialic acid also necessitates additional consideration of protective group compatibility in synthetic design.[22] Recently, it was discovered that the replacement of the 5-N-acetyl group on a sialyl donor with an electron withdrawing protective group[52–61] significantly enhanced the reaction yield and stereoselectivity. Thus, we designed sialyl donor 17 bearing the 5-N trichloroacetyl (TCA) moiety, as NH-TCA can be converted to acetamide under a variety of reaction conditions including hydrogenolysis, radical reduction and basic cleavage followed by acetylation.[62–64] The trifluoroacetimidate aglycon leaving group[58, 65, 66] was chosen due to the possibility for its selective activation over a thioglycoside. This was realized by treating a mixture of donor 17 and thiogalactoside acceptor 18[62–64] with a catalytic amount of TMSOTf leading to disaccharide 24 in 68% yield (Table 1, entry 1), which was benzoylated to give disaccharide 30. The α sialyl linkage was confirmed by the three bond coupling constant between C₁ and H_3ax (³J (C₁,H_3ax) = 5.8 Hz) of the sialic acid.[67] The scope of this sialylation reaction was examined next. A variety of acceptors including primary alkyl alcohol, carbohydrate hydroxyl groups of galactose, galactosamine and glucosamine were sialylated in good yields and stereoselectivities, which represented some of the common natural sialyl linkages (Table 1). Acid labile benzylidene and isopropylidene groups were stable under the reaction condition. In addition to acceptor 18, thioglycoside 23 also served as an excellent substrate for the reaction. This selective activation protocol is attractive as the resulting sialylated thioglycoside product can be used as a donor for further glycosylation without additional aglycon leaving group adjustment.

Table 1.

Sialylation Results using Sialyl Donor 17

Entry	Acceptor	Product	Yield (α : β)a)
1			68% (α only)
2			75 % (14:1)
3			91 % (33:1)
4			85 % (13:1)
5			85% (α only)
6			77% (α only)

^[a]Anomeric ratios were determined by ¹ H NMR analysis

With all building blocks in hand, one pot glycosylation was performed using the pre-activation based protocol (Scheme 3).[39, 44, 46, 68, 69] Pre-activation of disaccharide 30 by p-TolSCl/AgOTf,[44, 45] was rapidly achieved at −78 °C. Addition of the thioglycosyl acceptor 31[39] to the reaction mixture produced trisaccharide 3,[38] which underwent double glycosylation of hexasaccharide 4 in the same reaction flask leading to dodecasaccharide 2. It took only four hours for this three-component one pot assembly process and the desired dodecasaccharide 2 was easily isolated from the reaction by flash column chromatography in an excellent 65% yield from disaccharide 30.

Scheme 3.

Deprotection in complex oligosaccharide synthesis can be very challenging. Previously, Ito and coworkers reported that the removal of dichlorophthalimide (DCPhth) group in the presence of protected sialic acids did not lead to the desired product presumably due to cross reactivities of the methyl ester of sialic acids with reagents used to remove DCPhth and vice versa.[22] They designed an elegant approach by first converting all DCPhth groups into azide prior to the attachment of sialic acid, thus bypassing the cross reactivity problem. As an alternative, we explored the possibility of deprotecting sialic esters in the presence of phthalimide (Phth) groups. Treatment of the fully protected dodecasaccharide 2 with LiI in pyridine[54, 70] at 110 °C cleanly cleaved the two methyl esters without affecting the Phth groups (Scheme 4). The resulting dicarboxylic acid was then added to hydrazine in refluxing ethanol, which was followed by selective acetylation in methanol to yield compound 32. The usage of ethylene diamine instead of hydrazine to remove the Phth groups led to a lower yield. Finally, the fully deprotected dodecasaccharide 1 was produced by catalytic hydrogenolysis of 32 with Pd(OH)₂/C (49% overall yield from 2). The NMR and MS data of 1 are consistent with its structure.

Scheme 4.

Conclusion

We have achieved a stereocontrolled synthesis of the fucosylated complex type N glycan dodecasaccharide 2 via the pre-activation based chemoselective glycosylation method. This is the most complex oligosaccharide assembled by any one pot methods to date, which contains twelve monosaccharide units and several synthetically challenging linkages such as α-sialic acids, β-mannose, α-fucose as well as branching. Starting from monosaccharide building blocks, dodecasaccharide 2 was produced in a total of 10 steps with 7 steps and 11% overall yield for the longest linear sequence. The successful completion and high efficiency of this synthesis highlight the power of the pre-activation based approach. Due to the convergent modular approach taken, this strategy can be readily adapted towards the assembly of a library of N glycan sequences including bisecting GlcNAc and multi-antennary structures as well as un-natural N glycan analogs. We are currently synthesizing other N-glycan sequences and investigating the usage of these molecules as disease markers for early diagnosis.

Experimental Section

General Experimental Procedures

All reactions were carried out under nitrogen with anhydrous solvents in flame-dried glassware, unless otherwise noted. All glycosylation reactions were performed in the presence of molecular sieves, which were flame-dried right before the reaction under high vacuum. Glycosylation solvents were dried using a solvent purification system and used directly without further drying. Chemicals used were reagent grade as supplied except where noted. Analytical thin-layer chromatography was performed using silica gel 60 F254 glass plates; Compound spots were visualized by UV light (254 nm) and by staining with a yellow solution containing Ce(NH₄)₂(NO₃)₆ (0.5 g) and (NH₄)₆Mo₇O₂₄ 4H₂O (24.0 g) in 6% H₂SO₄ (500 mL). Flash column chromatography was performed on silica gel 60 (230–400 Mesh). NMR spectra were referenced using Me₄Si (0 ppm), residual CHCl₃ (δ ¹H-NMR 7.26 ppm, ¹³C-NMR 77.0 ppm). Peak and coupling constant assignments are based on ¹H-NMR, ¹H-¹H gCOSY and (or) ¹H-¹³C gHMQC and ¹H-¹³C gHMBC experiments. All optical rotations were measured using the sodium D line. ESI mass spectra were recorded using ESQUIRE Ion Trap LC/MS. High-resolution mass spectra were recorded on a Micromass electrospray mass spectrometer equipped with an orthogonal electrospray source (Z-spray) operated in positive ion mode.

Characterization of anomeric stereochemistry

The stereochemistries of the newly formed glycosidic linkages in oligosaccharides (except sialyl and mannosyl linkages) are determined by ³J (H₁, H₂) through ¹H-NMR and/or ¹J (C₁, H₁) through gHMQC 2-D NMR (without ¹H decoupling). Smaller coupling constants of ³J_H1.H2 (around 3 Hz) indicate 1,2-cis α linkages and larger coupling constants ³J (H₁,H₂) (7.2 Hz or larger) indicate 1,2-trans β linkages. This can be further confirmed by ¹J_C1,H1 (∼170 Hz) for α linkages and ¹J (C₁,H₁) (∼160 Hz) for β linkages.[47] For the mannosyl linkages, one bond coupling constants between C₁ and H₁ were measured by gHMQC 2-D NMR. ¹J (C₁, H₁) ∼170 Hz indicates α-mannosyl linkages and ¹J (C₁, H₁) ∼160 Hz suggests β linkages. For the sialyl linkages, three bond coupling constants between C₁ and H_3ax of the sialic acid was measured by gHMBC 2-D NMR. ¹J (C₁, H_3ax) ∼ 5.8 Hz indicates α-sialyl linkage and ¹J (C₁, H_3ax) ∼ 1Hz suggests β linkage.

Benzyl 2,3,4-tri-O-benzyl-α-L-fucopyranosyl-(1→6)-3-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (9)

A mixture of CuBr₂ (0.9 g, 6.27 mmol), Bu₄NBr (1.31 g, 4.06 mmol), and molecular sieves 4A (1.2 g) in CH₂Cl₂/DMF (2:1, 18 mL) was stirred and cooled over an ice-water bath. A solution of compound 7 (1.07 g, 1.98 mmol) and 8 (0.9 g, 1.84 mmol) in CH₂Cl₂ (12 mL) was added dropwise and the mixture was stirred for 17 h from 0°C to room temperature. The resulting mixture was quenched with aq. NaHCO₃, diluted with EtOAc, and filtered through Celite. The filtrate was washed successively with aq. NaHCO₃ and brine, and dried over Na₂SO₄. The organic layer was then evaporated in vacuo. The residue was purified by flush column chromatography (hexanes: EtOAc = 2:1) to afford 9 (1.25 g, 75%). [α]_D²⁰ = −78.1, (c = 1.0 in CHCl₃). ¹H NMR (600 Hz, CDCl₃): δ = 7.79 (bs, 1 H), 7.67 (bs, 2 H), 7.52 (bs, 1 H), 7.42-7.40 (m, 2 H), 7.38-7.22 (m, 14 H), 7.08-6.98 (m, 6 H), 6.89-6.86 (m, 3 H), 5.12-5.08 (m, 1 H), 4.98 (d, ³J (H, H) = 11.4 Hz, 1 H), 4.87 (d, ³J (H, H) = 11.4 Hz, 1 H), 4.84 (d, ³J (H, H)= 12 Hz, 1 H), 4.81 (d, ³J(H, H) = 3.6 Hz, 1 H), 4.78 (d, ³J = 12 Hz, 1 H), 4.74-4.70 (m, 2 H), 4.68 (d, ³J (H, H) = 11.4 Hz, 1 H), 4.65 (d, ³J (H, H) = 11.4 Hz, 1 H), 4.46 (d, ³J (H, H) = 12 Hz, 1 H), 4.41 (d, ³J (H, H) = 12.6 Hz, 1 H), 4.16-4.14 (m, 2 H), 4.08-4.06 (dd, ³J (H, H) = 3.6, 10.2 Hz, 1 H), 4.00-3.97 (m, 2 H), 3.94-3.90 (m, 2 H), 3.88-3.84 (dd, ³J (H, H) = 4.2, 10.8 Hz, 1 H), 3.70-3.67 (m, 2 H), 3.57-3.53 (m, 1 H), 1.12 (d, ³J ()H, H = 6.6 Hz, 3 H). ¹³C NMR (150 Hz, CDCl₃): δ = 138.80, 138.79, 138.7, 138.3, 137.3, 133.8, 131.9, 128.72, 128.66, 128.6, 128.52, 128.47, 128.34, 128.25, 128.24, 128.18, 128.1, 127.9, 127.83, 127.76, 127.5, 123.3, 98.9 (C₁ fucose, ¹J (C₁, H₁) = 172 Hz), 97.6 (C₁ glucosamine, ¹J (C₁, H₁) = 161 Hz), 79.5, 77.8, 77,7, 76.6, 75.1, 74.4, 74.4, 74.1, 73.2, 73.2, 60.6, 55.8, 16.7. Correlations between C₁ of fucose and H₆ and H_6’ of the glucosamine unit were observed in gHMBC 2-D NMR spectrum confirming the fucose is linked to 6-O of the glucosamine. HRMS: m/z: calcd for C₅₅H₅₅NO₁₁Na: [M+Na]⁺, 928.3673, found: 928.3654.

p-Tolyl 2-O-benzyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-1-thio-α-d-mannopyranoside (10α)

n-Bu₂SnO (3.72 g, 14.6 mmol) was added to a solution of p-tolyl 4,6-O-benzylidene-1-thio-α-d-mannopyranoside[71] (4.9 g, 13.6 mmol) in toluene (65 mL) and the resulting mixture was refluxed using a Dean-Stark apparatus for 3 h. The reaction mixture was cooled to room temperature and p-methoxybenzyl chloride (2.8 mL, 20.4 mmol) and n-Bu₄NI (1.01 g, 2.7 mmol) were added. The reaction mixture was refluxed again for 2 h followed by addition of H₂O (2 mL) to quench the reaction. After removing the solvents, the residue was purified by column chromatography (silica gel, hexanes: EtOAc 7 : 3) to give p-tolyl 4,6-O-benzylidene-3-O-p-methoxybenzyl-1-thio-α-d-mannopyranoside (5.96 g, 92%). This product was dissolved in anhydrous DMF (50 mL) and NaH (95%, 457 mg, 18 mmol) was added to the mixture. After 1 h at room temperature, benzyl bromide (2.47g 18 mmol) was added to the mixture and stirred overnight. The reaction mixture was diluted with EtOAc (300 mL), washed with water, 10% of NH₄Cl and brine and dried over anhydrous Na₂SO₄. The organic phase was evaporated and the residue was purified by flash column chromatography (silica gel, hexanes: EtOAc = 4 : 1) to give compound 10α (6.3 g, 90%). ¹H NMR (600 MHz, CDCl₃): δ = 7.58-7.53 (m, 2 H), 7.44-7.27 (m, 12 H), 7.15-7.11 (m, 2 H), 6.93-6.87 (m, 2 H), 5.67 (s, 1 H), 5.46 (d, ³J (H, H) = 1.8 Hz, 1 H), 4.76 (d, ³J (H, H) = 12 Hz, 1 H), 4.73 (s, 2 H), 4.62 (d, ³J (H, H) = 12 Hz, 1 H), 4.36-4.30 (m, 2 H), 4.27-4.22 (dd, ³J (H, H) = 10.8, 4.2 Hz, 1 H), 4.40-4.10 (m, 1 H), 4.10-3.97 (m, 1 H), 3.94- 3.88 (m, 1 H), 3.82 (s, 3 H), 2.35 (s, 3 H); ¹³C NMR (150 MHz, CDCl₃): δ =159.5, 138.2, 138.1, 137.9, 132.6, 130.7, 130.2, 130.16, 129.6, 129.1, 128.7, 128.5, 128.4, 128.1, 126.4, 114.0, 101.7, 87.7, 79.3, 78.2, 76.0, 73.2, 73.0, 68.8, 65.8, 21.4. HRMS: m/z calcd for C₃₅H₃₆O₆SNa, [M+Na]⁺, 607.2130, found 607.2089.

p-Tolyl 2-O-benzyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (12β)

A mixture of donor 10α (226 mg, 387 µmol) and TTBP (180 mg, 726 µmol) in dry CH₂Cl₂ (20 mL) was stirred with activated molecular sieves 4Å (800 mg) for 30 minutes at room temperature and then cooled to −75°C. AgOTf (118 mg, 0.46 mmol) in acetonitrile (300 µL) was added directly to the solution without touching the flask wall. After 10 minutes, orange colored p-TolSCl (55 µl, 387 µmol) was added via a microsyringe. Since the reaction temperature was lower than the freezing point of p-TolSCl, p-TolSCl was added directly into the reaction mixture to prevent it from freezing on the flask wall. The characteristic yellow color of p-TolSCl in the reaction solution dissipated rapidly within a few seconds indicating depletion of p-TolSCl. After the donor was completely consumed according to TLC analysis (about 5 minutes at −78 °C), a solution of acceptor 11 (345 mg, 579 µmol) in CH₂Cl₂ (6 mL) was added slowly along the flask wall. The reaction mixture was allowed to warm up to −20°C and quenched by using triethylamine. The reaction mixture was diluted with CH₂Cl₂ and filterer through Celite. The Celite was washed extensively with CH₂Cl₂ until TLC indicated no more organic compounds in the filtrate. The organic layer was then extracted with satd. NaHCO₃ solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. After flash column chromatography (hexanes : EtOAc = 7 : 3), the α and β mixture (231 mg, 82% based on consumption donor, recover donor 70 mg) was obtained. The pure 12α (33 mg) and 12β (198 mg) (α:β = 1:6) were isolated by column chromatography using 3% acetone in toluene. [α]_D²⁰ = +19.4, (c = 1.0 in CHCl₃). Data for 12β: ¹H NMR (600 MHz, CDCl₃): δ = 7.83-7.80 (m, 1H), 7.71-7.64 (m, 2H), 7.63-7.60 (m, 1H), 7.48-7.42 (m, 4H), 7.38-7.30 (m, 7H), 7.29-7.21 (m, 8H), 7.01-6.98 (m, 2H), 6.92-6.89 (m, 2H), 6.86-6.80 (m, 5H), 5.51 (s, 1H), 5.44 (d, ³J (H, H) = 10.2 Hz, 1 H), 4.90-4.82 (m, 3H), 4.65 (d, ³J (H, H) = 12 Hz, 1 H), 4.58 (d, ³J (H, H) = 12 Hz, 1 H), 4.55 (s, 1H), 4.51 (d, ³J (H, H) = 12 Hz, 1 H), 4.40 (t, 2H, J = 12.6 Hz), 4.25-4.22 (m, 1H), 4.19-4.15 (m, 2H), 4.05 (t, 1H, J = 9.6 Hz), 3.96 (t, 1H, J = 9.6 Hz), 3.78 (s, 3H), 3.75 (d, ³J (H, H)= 3 Hz, 1 H), 3.68 (d, ³J (H, H) = 12 Hz, 1 H), 3.59-3.51 (m, 3H), 3.43 (dd, 1H, J = 3.0, 10.2 Hz), 3.16-3.12 (m, 1H), 2.27 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ = 168.4, 167.5, 159.4, 138.9, 138.8, 138.5, 138.2, 137.9, 134.1, 134.1, 133.9, 133.7, 131.9, 131.8, 130.8, 129.8, 129.4, 129.3, 129.1, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.2, 126.3, 123.7, 123.5, 113.9, 102.3 (C₁ mannose, ¹J (C₁, H₁) = 154 Hz), 101.6, 83.4 (C₁ glucosamine, ¹J (C₁, H₁) = 157 Hz), 79.5, 79.4, 78.9, 78.2, 77.3, 75.2, 75.1, 73.8, 72.6, 68.9, 68.8, 67.6, 55.5, 55.0, 21.4; HRMS: m/z: calcd for C₆₃H₆₁NNaO₁₂S [M+Na]⁺: 1078.3812, found: 1078.3807.

p-Tolyl 2,4-di-O-benzyl-4,6-di-O-levulinoyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (14)

A solution of compound 12β (210 mg, 0.20 mmol) in CH₂Cl₂ (6 mL) was stirred with activated molecular sieves (MS AW 300) (600 mg) and cooled to −78°C. Triethylsilane (130 µL, 0.20 mmol) was added followed by dichlorophenyl borane (105 µL, 0.20 mmol). The reaction was stirred for 6 h and quenched by adding triethyl amine. The reaction mass was diluted with CH₂Cl₂ and extracted with a saturated aqueous NaHCO₃ solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Diol p-tolyl 2,4-di-O-benzyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (167 mg) was obtained from flash column chromatography (EtOAc: hexanes, 1: 1) in 89% yield. [α]_D²⁰ = + 40.6, (c = 1.0 in CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 7.83-7.80 (m, 1H), 7.71-7.61 (m, 3H), 7.40-7.25 (m, 16H), 7.04-7.01 (m, 2H), 6.92-6.88 (m, 5H), 5.43 (d, ³J (H, H) = 10.2 Hz, 1 H), 4.98 (d, ³J (H, H) = 11.4 Hz, 1 H), 4.86 (d, ³J (H, H) = 12.6 Hz, 1 H), 4.81-4.79 (d, ³J (H, H) = 10.8 Hz, 1 H), 4.66-4.58 (m, 3H), 4.53 (d, ³J (H, H) = 11.4 Hz, 1 H), 4.52-4.48 (d, ³J (H, H) = 12 Hz, 1 H), 4.25-4.17 (m, 2H), 3.99-3.96 (t, ³J (H, H) = 8.4 Hz, 1 H), 3.80-3.69 (m, 3H), 3.64-3.61 (m, 2H), 3.51-3.47 (m, 1H), 3.44-3.39 (m, 2H), 3.14-3.11 (m, 1H), 2.28 (s, 3H), 1.86-1.83 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ = 168.5, 167.9, 138.8, 138.6, 137.8, 137.5, 136.6, 136.2, 134.3, 134.2, 134.1, 133.9, 133.2, 132.1, 131.9, 131.6, 129.9, 128.8, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.3, 123.9, 123.5, 123.3, 96.8, 96.7, 78.776.3, 75.5, 74.7, 74.6, 74.4, 74.0, 73.6, 70.9, 69.9, 62.5, 60.7, 56.5, 56.1, 27.2; ESI-MS: m/z: calcd for C₅₅H₅₅NO₁₁SNa: [M+Na]⁺, 960.35, found: 960.36. To a solution of p-tolyl 2,4-di-O-benzyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (167 mg, 0.178 mmol) in CH₂Cl₂ (5 mL), levulinic acid (73 µL, 0.712 mmol), N-ethyl N,N-dimethylaminopropyl carbodiimide hydrochloride (EDC) (136.5 mg, 0.712 mmol) and N,N-dimethylamino pyridine (4.4 mg, 0.036 mmol) were added and the reaction was stirred overnight at room temperature. The reaction mixture was diluted with CH₂Cl₂ (50 mL) and extracted with satd. NaHCO₃ solution. The organic layer was evaporated to dryness and compound 14 (173 mg) was obtained in 86 % yield after flash column chromatographic purification (hexanes : EtOAc = 2:1). [α]_D²⁰ = +17.8, (c = 1.0 in CHCl₃). ¹H NMR (600 Hz, CDCl₃): δ = 7.78-7.74 (m, 1 H), 7.68-7.62 (m, 2H), 7.64-7.61 (m, 1 H), 7.42-7.24 (m, 17 H), 6.99-6.97 (m, 1 H), 6.83-6.81 (m, 2 H), 6.73-6.69 (m, 3 H), 5.39 (d, ³J (H, H) = 10.2 Hz, 1 H), 4.85 (d, ³J (H, H) = 12 Hz, 1H), 4.80 (d, ³J (H, H) = 12 Hz, 1 H), 4.77 (dd, ³J (H, H) = 3.0, 9.6 Hz, 1 H), 4.70 (d, ³J (H, H) = 12 Hz, 1 H), 4.67 (s, 2 H), 4.54 (d, ³J (H, H) = 12 Hz, 1 H), 4.53 (d, ³J (H, H) = 12 Hz, 1 H), 4.42 (d, ³J (H, H) = 12.6 Hz, 1 H), 4.32 (dd, ³J (H, H) = 4.8, 12 Hz, 1 H), 4.24-4.10 (m, 4 H), 4.02 (t, ³J (H, H)= 9.6 Hz, 1 H), 3.92 (d, ³J (H, H) = 3.0 Hz, 1 H), 3.87 (t, ³J (H, H) = 9.6 Hz, 1 H), 3.77 (d, ³J (H, H) = 10.8 Hz, 1 H), 3.68 (dd, ³J (H, H) = 3.0, 12 Hz, 1 H), 3.55 (d, ³J (H, H) =.6 Hz, 1 H), 3.38-3.33 (m, 1 H), 2.67-2.33 (m, 8 H), 2.25 (s, 3 H), 2.12 (s, 3 H), 2.01 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃): δ = 206.7, 206.2, 172.3, 172.0, 167.9, 167.2, 138.7, 138.6, 138.2, 137.89, 137.85, 133.8, 133.6, 129.5, 128.5, 128.4, 128.2, 127.94, 127.89, 127.78, 127.75, 127.71, 127.66, 127.51, 126.8, 123.3, 123.2, 100.7, 83.18, 79.0, 78.9, 77.9, 76.2, 76.1, 74.8, 74.7, 74.6, 73.4, 73.07, 73.05, 68.5, 63.1, 54.7, 37.74, 37.69, 29.8, 29.7, 27.88, 27.85, 21.1. MALDI-MS: m/z: calcd for C₆₅H₆₇NO₁₅SNa: [M+Na]⁺, 1156.42, found: 1156.61.

Benzyl 2,4-di-O-benzyl-3,6-di-O-levulinoyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→4)-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6))-3-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (15)

The mixture of donor 14 (100 mg, 88 µmol), acceptor 9 (64 mg, 70.4 µmol) and freshly activated MS 4Å (600 mg) in dry CH₂Cl₂ (6 mL) was stirred for 30 min at room temperature, and cooled down to −70°C followed by the addition of AgOTf (68 mg, 264 µmol) in anhydrous acetonitrile (0.1 mL) directly to the solution without touching the wall of reaction flask. After 5 min, p-TolSCl (12.8 µL, 88 µmol) was added via a microsyringe. The reaction mixture was stirred for 1.5 h until the temperature reached −20°C and triethylamine (30 µL) was added. The mixture was diluted with CH₂Cl₂ (50 mL) filtered through Celite. The filtrate was concentrated and purified by flash column chromatography (hexanes : EtOAc = 3 : 2) to give compound 15 (116 mg, 86%). [α]_D²⁰ = −28.0 (c = 1.0 in CHCl₃). ¹H NMR (600 Hz, CDCl₃): δ = 7.85-7.81 (m, 1 H), 7.80-7.76 (m, 1 H), 7.72-7.60 (m, 4H), 7.59-7.56 (m, 1H), 7.52-7.40 (m, 6H), 7.36-7.20 (m, 18H), 7.17-7.12 (m, 3H), 7.05-7.02 (m, 3 H), 6.98-6.94 (m, 4 H), 6.92-6.88 (m, 2 H), 6.80-6.71 (m, 8 H), 5.52 (d, 1 H, J = 8.4 Hz), 4.92-4.70 (m, 10H), 4.68 (s, 1 H), 4.66-4.47 (m, 8 H), 4.39 (d, ³J (H, H) = 12.6 Hz, 1 H), 4.33 (d, ³J (H, H) = 12 Hz, 1 H), 4.30-4.10 (m, 8 H), 4.09-4.04 (m, 1 H), 3.95-3.92 (m, 3 H), 3.84-3.76 (m, 4 H), 3.68 (d, ³J (H, H) = 9.0 Hz, 1 H), 3.60-3.56 (m, 2 H), 3.37-3.30 (m, 2 H), 3.22 (d, ³J (H, H) = 9.6 Hz, 1 H), 2.62-2.27 (m, 8 H), 2.10 (s, 3 H), 1.93 (s, 3 H), 0.95 (d, ³J (H, H) = 5.4 Hz, 3 H). ¹³C NMR (150 Hz, CDCl₃): δ = 206.9, 206.4, 172.5, 172.3, 168.4, 168.0, 167.94, 167.85, 139.3, 139.2, 139.1, 139.00, 138.86, 138.85, 138.2, 138.1, 137.3, 134.1, 133.9, 132.1, 131.9, 131.8, 128.9, 128.8, 128.68, 128.67, 128.6, 128.40, 128.35, 128.3, 128.13, 128.11, 128.08, 128.04, 128.00, 127.99, 127.9, 127.81, 127.77, 127.74, 127.71, 127.6, 127.50, 127.48, 127.2, 127.1, 123.7, 123.5, 100.8, 97.1, 96.92, 96.85, 79.8, 79.2, 77.9, 77.1, 76.9, 76.5, 76.2, 75.4, 75.3, 75.0, 74.96, 74.7, 74.6, 74.6, 73.9, 73.5, 73.46, 73.3, 73.28, 72.7, 70.1, 68.2, 66.2, 64.0, 63.3, 60.6, 56.8, 56.1, 38.0, 37.99, 30.0, 29.9, 28.2, 28.1, 16.6. HRMS: m/z: calcd for C₁₁₃H₁₁₄N₂O₂₆Na, [M+Na]⁺, 1938.7592, found: 1938.7534.

Benzyl 2,4-di-O-benzyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→4)-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6))-3-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (6)

To a solution of 15 (300 mg, 0.157 mmol) in CH₂Cl₂/MeOH (1:1, 8 mL), hydrazine acetate (60 mg, 0.65 mmol) was added and stirred at room temperature for 90 min. The reaction mixture was diluted with CH₂Cl₂ (100 mL) and extracted with satd. NH₄Cl solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The compound 6 (217 mg, 81%) was isolated by flash column chromatography (hexanes: EtOAc = 2 : 3). [α]_D²⁰ = −16.5 (c = 1.0 in CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 7.92-7.88 (m, 1 H), 7.84-7.78 (m, 1 H), 7.76-7.62 (m, 5 H), 7.58-7.55 (m, 1H), 7.52-7.42 (m, 1H), 7.40-7.22 (m, 22H), 7.08-7.00 (m, 7H), 6.98-6.90 (m 7H), 6.81-6.76 (m, 3H), 5.64 (d, ³J (H, H) = 8.4 Hz, 1 H), 5.00-4.90 (m, 7 H), 4.86-4.76 (m, 4 H), 4.67-4.60 (m, 5 H), 4.57-4.51 (m, 2 H), 4.46-4.26 (m, 6 H), 4.24-4.16 (m, 3 H), 4.10-4.00 (m, 3 H), 3.93 (d, ³J (H, H) = 10.2 Hz, 1 H), 3.86 (d, ³J (H, H) = 9.6 Hz, 1 H), 3.77 (d, ³J (H, H) = 10.2 Hz, 1 H), 3.75 (d, ³J (H, H) = 11.4 Hz, 1 H), 3.69-3.64 (m, 2 H), 3.60 (d, ³J (H, H) = 3.6 Hz, 1 H), 3.55-3.49 (m, 1 H), 3.45-3.40 (m, 3 H), 3.29 (d, ³J (H, H)= 9.6 Hz, 1 H), 3.18-3.13 (m, 1 H), 2.34 (d, ³J (H, H) = 9.0 Hz, 1 H), 1.02 (d, ³J (H, H) = 6.6 Hz, 3 H). ¹³C NMR (150 MHz, CDCl₃): δ = 168.7, 168.1, 168.0, 167.9, 139.2, 139.1, 139.0, 138.9, 138.8, 138.5, 138.3, 1378.0, 137.3, 134.3, 134.1, 133.9, 132.1, 132.0, 131.7, 128.9, 128.74, 128.69, 128.66, 128.64, 128.61, 128.4, 128.38, 128.3, 128.26, 128.2, 128.1, 128.08, 128.0, 127.94, 127.81, 127.8, 127.77, 127.7, 127.64, 127.6, 127.4, 127.2, 123.9, 123.5, 101.4, 97.0, 96.99, 96.9, 79.8, 79.2, 79.0, 77.8, 77.4, 76.8, 76.2, 75.4, 75.37, 75.36, 75.3, 75.0, 74.9, 74.7, 74.67, 74.4, 73.7, 73.69, 73.4, 72.7, 70.2, 68.1, 66.3, 64.0, 62.6, 60.7, 56.7, 56.1, 16.6. HRMS: m/z: calcd for [M+Na]⁺, C₁₀₃H₁₀₂N₂O₂₂Na, 1742.6855, found: 1742.6771.

Benzyl 2-O-acetyl-3,4,6-tri-O-benzyl-α-d-mannopyranosyl-(1→6)-(2-O-acetyl-3,4,6-tri-O-benzyl-α-d-mannopyranosyl-(1→3))-2,4-di-O-benzyl-β-d-mannopyranosyl-(1→4)-O-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→4)-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6))-3-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (16)

The mixture of donor 5 (26 mg, 45 µmol), acceptor 6 (17 mg, 15.7 µmol) and freshly activated MS 4Å (600 mg) in anhydrous CH₂Cl₂ (6 mL) was stirred for 30 minutes at room temperature and cooled down to −78°C followed by the addition of AgOTf (34 mg, 133 µmol) in anhydrous acetonitrile (0.1 mL) directly to the solution without touching the wall of reaction flask. After 5 min, p-TolSCl (6.8 µL, 45 µmol) was added via a microsyringe. The reaction mixture was stirred for 1.5 h until the temperature reached 0°C, which was followed by addition of TMSOTf (2 µL). The reaction was stirred further for 1.5 h from 0°C to room temperature and triethylamine (25 µL) was added. The mixture was diluted with CH₂Cl₂ (50 mL) and filtered through Celite. The Celite was washed with CH₂Cl₂ until no organic product was present in the filtrate by TLC. The filtrate was combined, concentrated and purified by flash column chromatography (hexanes : EtOAc = 3 : 2) to give compound 16 (32 mg, 76%). [α]_D²⁰ = + 4.5 (c = 1.0 in CHCl₃). ¹H NMR (600 Hz, CDCl₃): δ = 7.76-7.71 (m, 2 H), 7.69-7.58 (m, 4 H), 7.50-7.45 (m, 4 H), 7.41-7.16 (m, 42 H), 7.14-7.01 (m, 11 H), 6.99-6.93 (m, 4 H), 6.93-6.88 (m, 2 H), 6.78-6.68 (m, 6 H), 6.62-6.58 (m, 2 H), 5.49-5.46 (m, 2 H), 5.30-5.28 (m, 1 H), 5.14 (s, 1 H), 4.93 (d, ³J (H, H) = 8.4 Hz, 1 H), 4.92-4.80 (m, 10 H), 4.77 (d, ³J (H, H)= 10.8 Hz, 1 H), 4.71 (d, ³J (H, H)= 11.4 Hz, 1 H), 4.67 (d, ³J (H, H)= 11.4 Hz, 1 H), 4.64-4.52 (m, 9 H), 4.50-4.43 (m, 4 H), 4.42-4.33 (m, 5 H), 4.29 (d, ³J (H, H)= 12.6 Hz, 1 H), 4.28-4.12 (m, 6 H), 4.10-4.04 (m, 2 H), 3.99-3.87 (m, 6 H), 3.85-3.70 (m, 7 H), 3.67-3.56 (m, 7 H), 3.5-3.48 (m, 2 H), 3.32-3.28 (dd, ³J (H, H) = 3.0, 10.2 Hz, 1 H), 3.21 (d, ³J (H, H) = 9.6 Hz, 1 H), 3.17-3.13 (m, 1 H), 2.10 (s, 3 H), 1.80 (s, 3 H), 0.96 (d, ³J (H, H)= 6.6 Hz, 3 H). ¹³C NMR (150 Hz, CDCl₃): δ = 170.4, 169.9, 168.8, 167.78, 139.2, 139.0, 138.9, 138.85, 138.6, 138.3, 138.2, 138.15, 137.8, 137.7, 137.3, 133.9, 133.7, 132.1, 131.9, 131.7, 128.9, 128.7, 128.61, 128.5, 128.4, 128.3, 128.1, 128.0, 127.8, 127.7, 127.6, 127.4, 127.3, 127.1, 127.0, 123.7, 123.4, 102.0, 99.7, 99.6, 98.5, 97.1, 96.8, 81.5, 79.8, 79.5, 78.9, 78.4, 77.98, 77.9, 76.7, 76.4, 75.7, 75.3, 75.2, 75.19, 75.0, 74.9, 74.85, 74.8, 74.7, 74.69, 74.67, 74.6, 74.5, 74.4, 74.2, 74.0, 73.7, 73.6, 73.5, 73.46, 72.6, 72.5, 72.0, 71.6, 71.4, 70.1, 70.0, 69.0, 68.9, 68.34, 68.129, 66.7, 66.2, 65.3, 64.0, 56.8, 56.0, 24.9, 21.3, 21.2, 16.7. MALDI-MS: m/z: calcd for C₁₆₁H₁₆₂N₂O₃₄Na, [M+Na]⁺, 2690.10, found: 2690.49.

Benzyl 3,4,6-tri-O-benzyl-α-d-mannopyranosyl-(1→6)-(3,4,6-tri-O-benzyl-α-d-mannopyranosyl-(1→3))-2,4-di-O-benzyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→4)-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6))-3-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (4)

The solution of compound 16 (242 mg, 90.6 µmol) in methanol (5 mL) and CH₂Cl₂ (2 mL) was stirred with NaOMe (12 mg, 0.5 mmol) at room temperature for 3 h, which was then neutralized to pH 5-6 with acetic acid. After evaporating the solvent, the residue was purified by flash column chromatography (hexanes: EtOAc = 1 : 1) to give 4 (184 mg, 79%). [α]_D²⁰ = +4.0 (c = 1.0 in CHCl₃). ¹H NMR (600 Hz, CDCl₃): δ = 7.77-7.83 (m, 2 H), 7.72-7.61 (m, 4 H), 7.56-7.47 (m, 4 H), 7.45-7.39 (m, 4 H), 7.36-7.10 (m, 53 H), 7.09-7.03 (m, 2 H), 7.02-6.97 (m, 4 H), 6.94-6.91 (m, 2 H), 6.80-6.73 (m, 6 H), 6.70-6.66 (m, 2 H), 5.52 (d, 1 H, J = 8.4 Hz), 5.19 (s, 1 H), 5.00-4.82 (m, 11 H), 4.79-4.73 (m, 2 H), 4.67-4.45 (m, 15 H), 4.45-4.37 (m, 3 H), 4.36-4.23 (m, 6 H), 4.21-4.10 (m, 4 H), 4.04-3.96 (m, 4 H), 3.92-3.50 (m, 20 H), 3.35 (dd, ³J (H, H) = 2.4, 10.8 Hz, 1 H), 3.28 (d, ³J (H, H)= 9.6 Hz, 1 H), 3.21 (d, ³J (H, H)= 9.6 Hz, 1 H), 2.38 (bs, 1 H), 2.07 (bs, 1 H), 0.99 (d, ³J (H, H) = 6.6 Hz, 3 H). ¹³C NMR (150 Hz, CDCl₃): δ = 168.3, 168.0, 139.2, 139.2, 139.15, 139.1, 138.9, 138.8, 138.75, 138.6, 138.4, 138.3, 138.2, 138.16, 138.1, 137.3, 134.1, 133.8, 132.1, 131.7, 128.9, 128.73, 128.70, 128.69, 128.67, 128.6, 128.5, 128.48, 128.46, 128.4, 128.3, 128.2, 128.16, 128.1, 128.09, 128.07, 128.04, 128.02, 127.98, 127.95, 127.93, 127.89, 127.85, 127.8, 127.76, 127.74, 127.72, 127.67, 127.6, 127.57, 127.3, 127.1, 127.06, 123.7, 123.4, 101.8, 101.5, 100.1, 97.2, 97.1, 96.8, 81.9, 80.3, 80.0, 79.56, 79.3, 78.8, 77.9, 76.5, 75.8, 75.5, 75.3, 75.1, 75.07, 75.0, 74.8, 74.75, 74.7, 74.6, 74.5, 74.4, 74.3, 74.0, 73.7, 73.5, 73.47, 72.7, 72.3, 72.2, 71.8, 71.4, 70.1, 69.0, 68.9, 68.2, 67.9, 66.7, 66.2, 64.0, 56.8, 56.1, 16.7. HRMS: m/z: calcd for C₁₅₇H₁₅₈N₂O₃₂Na, [M+Na]⁺, 2607.0730, found: 2607.0669.

Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-α-d-galacto-2-nonulopyranosonate-2-(N-phenyl)-trifluoroacetimidate (17)

To a solution of methyl (p-tolyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-d-glycero-d-galacto-2-nonulopyranosid)onate (1.8 g, 3 mmol)[67] in methanol (30 mL) was added methanesulfonic acid (1.95 mL, 30 mmol) at room temperature. After being stirred at 60°C for 24 h, the reaction mixture was neutralized with triethylamine and concentrated in vacuo. The residue was used for next reaction without further purification. Methyl trichloroacetate (3.57 mL, 30 mmol) and triethylamine (0.84 mL, 6 mmol) were added to a solution of the residue in methanol (30 mL) at 0°C. The reaction mixture was stirred at room temperature for 6 h and all volatile solvents were removed. The remaining residue was dissolved in pyridine (10 mL), to which acetic anhydride (2 mL) and a catalytic amount of DMAP were added at 0°C. After being stirred at room temperature overnight, the reaction mixture was diluted with ethyl acetate, washed with water, 1 M HCl, saturated aq. NaHCO₃, and brine, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc : hexanes = 2 : 3) to give methyl (p-tolyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-5-trichloroacetamido-D-glycero-D-galacto-2-nonulopyranosid)onate (1.40 g, 2 mmol, 67%). To a solution of this thioglycoside (1.4 g, 2 mmol) in acetone (20 mL) and water (1 mL) was added NBS (930 mg, 5.23 mmol).[72] After being stirred for 0.5 h at room temperature, the solution was concentrated. The residue was diluted with CH₂Cl₂ and washed with saturated aq. NaHCO₃. The organic layer was dried over Na₂SO₄ and concentrated leading to methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-β-d-glycero-d-galacto-2-nonulopyranosonate. To a solution of methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-d-galacto-2-nonulopyranosonate in acetone was added K₂CO₃ (3 eq) and N-phenyl trifluoroacetimidoyl chloride[66] (10 eq). After stirred at room temperature for 3 h, the mixture was filtered and then concentrated. The residue was subject to chromatography on silica gel column (hexanes: EtOAc = 3 : 2, containing 0.5% of triethylamine) leading to imidate 17 (α : β= 1 : 2, 79%). α : β mixture ¹H NMR (400 MHz, CDCl₃): δ = 7.32-7.25 (m, 1.5 H), 7.15-7.08 (m, 1.5 H), 6.83-6.77 (m, 1.5 H), 6.76-6.61 (m, 3 H), 5.50-5.47 (m, 1 H, β), 5.46-5.40 (m, 1 H, β), 5.36-5.33 (m, 0.5 H, α), 5.33-5.28 (m, 0.5 H, α), 5.18-5.13 (m, 1 H, β), 4.91-4.84 (m, 0.5 H, α), 4.55-4.49 (m, 1 H, β), 4.46-4.41 (m, 1 H, β), 4.38-4.32 (m, 0.5 H, α), 4.29-4.23 (m, 0.5 H, α), 4.18-4.12 (m, 2 H, β), 4.10-3.97 (m, 1 H, α), 2.94-2.87 (dd, ³J (H, H) = 13.6, 4.8 Hz, β, H-3eq, 1 H), 2.83-2.77 (dd, ³J (H, H) = 4.8, 13.6 Hz, 3-Heq,α, 0.5 H), 2.38-2.30 (dd, ³J (H, H)= 10.8, 13.6 Hz, 3-H_ax,α, 0.5 H), 2.26-2.18 (dd, ³J (H, H) = 11.6, 13.6 Hz, 3-Hax, β 1 H), 2.20 (s, 1.5 H, α), 2.19 (s, 3 H, β), 2.09, 2.05, 1.84, 1.62 (each 3 H, each s, OAc from β isomer), 2.04 (s, 3 H, from 2 OAc of α isomer), 1.98 (s, 1.5 H, α), 1.96 (1.5 H, α). HRMS: m/z calcd for C₂₈H₃₀Cl₃F₃N₂O₁₃Na, [M+Na]⁺, 787.0663, found 787.0626.

p-Tolyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-α-d-galacto-2-nonulopyranosonate-(2→3))-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (24)

A mixture of the donor 17 (514 mg, 0.67 mmol) and acceptor 18 (411 mg, 1.1 mmol) and MS 3Å in anhydrous CH₂Cl₂/CH₃CN (1 : 1, 30 mL) was stirred at room temperature under N₂ for 30 min and then cooled to −65°C. TMSOTf (0.2 eq) was added. After stirred at −65°C for 2 h, the mixture was warmed up to room temperature and quenched with triethylamine (50 µL). The resulting mixture was filtered and concentrated. The residue was chromatographed on a silica gel column to afford the desired coupling product 24 (346 mg, 68% based on acceptor consumed, recover acceptor 210 mg). [α]_D²⁰ = −11.2, (c = 1.0 in CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 7.61-7.57 (m, 2 H), 7.38-7.30 (m, 5 H), 7.07-7.04 (m, 2 H), 6.52 (d, ³J (H, H) = 9.6 Hz, 1 H), 5.43-5.39 (m, 1 H), 5.32 (s, 1 H), 5.27-5.24 (dd, 1 H, J = 1.8, 9.6 Hz), 5.11-5.05 (m, 1 H), 4.63 (d, ³J (H, H) = 9.6 Hz, 1 H), 4.32 (dd, ³J (H, H) = 1.2, 12 Hz, 1 H), 4.29-4.24 (m, 2 H), 4.22 (dd, ³J (H, H) = 1.8, 10.8 Hz, 1 H), 4.10-4.05 (m, 2H), 3.97 (d, ³J (H, H)= 3.0 Hz, 1 H), 3.82 (q, ³J (H, H) = 10.2 Hz, 1 H), 3.76 (dt, ³J (H, H) = 0.6, 9.6 Hz, 1 H), 3.58 (s, 3 H), 3.54 (s, 1 H), 2.72 (dd, ³J (H, H) = 4.2, 12.6 Hz, 1 H), 2.58 (d, ³J (H, H) = 1.8 Hz, 1 H), 2.32 (s, 3 H), 2.19 (s, 3 H), 2.18 (s, 3 H), 2.00 (s, 3 H), 1.98 (s, 3 H), 1.94 (dd, ³J (H, H) = 6.0, 12.6 Hz, 1 H). ¹³C NMR (150 MHz, CDCl₃): δ = 170.9, 170.7, 170.5, 170.4, 168.4, 162.3, 138.2, 138.15, 134.42, 134.41, 129.81, 129.77, 129.2, 128.3, 127.4, 126.8, 101.1, 97.4, 92.3, 86.8, 76.4, 74.0, 71.9, 69.7, 69.5, 68.1, 67.6, 67.3, 66.0, 62.5, 53.3, 51.8, 38.7, 21.6, 21.5, 21.04, 21.01, 20.97. HRMS: m/z, calcd for C₄₀H₄₆Cl₃NO₁₇SNa, [M+Na]⁺, 972.1444, found: 972.1448. The sialyl α2→3 galactose linkage was confirmed by a HMBC correlation between C₂ of the sialic acid and H₃ of the galactose unit. Stereochemistry of the linkage was established using compound 30.

p-Tolyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-α-d-galacto-2-nonulopyranosonate-(2→3))-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (30)

The mixture of compound 24 (475 mg, 0.5 mmol), pyridine (2 mL) and benzoyl chloride (1 mL) was stirred overnight at room temperature. The mixture was then diluted with CH₂Cl₂ (100 mL) and washed with HCl (1 M), water and satd. sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. After flash column chromatography (EtOAc:CH₂Cl₂:hexanes = 3 : 3 : 4), compound 30 was obtained (501 mg, 95%). [α]_D²⁰= −15.0, (c = 1.0 in CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 8.16-8.12 (m, 2 H), 7.60-7.56 (m, 1H), 7.52-7.46 (m, 2 H), 7.45-7.42 (m, 2 H), 7.39-7.35 (m, 2 H), 7.35-7.30 (m, 3 H), 7.02-7.00 (m, 2 H), 6.58 (d, ³J (H, H) = 10.2 Hz, 1 H), 5.49-5.44 (m, 1 H), 5.33 (t, ³J (H, H) = 9.6 Hz, 1 H), 5.32 (s, 1 H), 5.18 (dd, ³J (H, H) = 1.8, 9.6 Hz, 1 H), 4.92-4.86 (m, 2 H), 4.56 (dd, ³J (H, H) = 3.6, 9.6 Hz, 1 H), 4.34 (d, ³J (H, H) = 11.4 Hz, 1 H), 4.30 (dd, ³J (H, H) = 2.4, 6.6 Hz, 1 H), 4.10-4.03 (m, 2 H), 4.02-3.96 (m, 2 H), 3.72 (q, ³J (H, H) = 10.2 Hz, 1 H), 3.62 (s, 1 H), 3.54 (s, 3 H), 2.58 (dd, ³J (H, H) = 4.8, 13.2 Hz, 1 H), 2.29 (s, 3 H), 2.18 (s, 3 H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.76 (s, 3 H), 1.64 (t, ³J (H, H) = 13.2 Hz, 1 H). ¹³C NMR (150 MHz, CDCl₃): δ = 170.9, 170.6, 170.1, 168.8, 165.1, 162.3, 138.4, 138.0, 134.6, 133.3, 130.8, 130.7, 130.2, 129.7, 129.3, 129.2, 128.7, 128.3, 127.7, 126.8, 101.3, 96.9, 92.2, 85.5, 73.73, 73.68, 71.9, 69.7, 69.5, 68.5, 68.0, 67.9, 67.2, 62.7, 53.2, 51.5, 38.5, 21.7, 21.5, 21.01, 20.93, 20.6. HRMS: m/z: calcd for C₄₇H₅₀Cl₃NO₁₈SNa, [M+Na]⁺, 1076.1712, found: 1076.1676. Three-bond coupling between CO₂Me and H_3ax of sialic acid was determined to be 5.8 Hz indicating α sialyl linkage.

Benzyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-α-d-galacto-2-nonulopyranosonate-(2→3))-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside-(1→2)-3,4,6-tri-O-benzyl-α-d-mannopyranosyl-(1→6)-((methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-α-d-galacto-2-nonulopyranosonate-(2→3))-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside-(1→2)-3,4,6-tri-O-benzyl-α-d-mannopyranosyl-(1→3))-2,4-di-O-benzyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→4)-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6))-3-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside 2

A mixture of donor 30 (53 mg, 0.05 mmol) and freshly activated molecular sieves 4Å (800 mg) in CH₂Cl₂ (6 mL) was stirred at room temperature for 30 minutes and then cooled to −78 °C, which was followed by the addition of AgOTf (38 mg, 0.15 mmol) dissolved in acetonitrile (0.1 mL) without touching the wall of the flask. After 5 minutes, orange colored p-TolSCl (7.6 µL, 0.05 mmol) was added through a microsyringe. Since the reaction temperature was lower than the freezing point of p-TolSCl, p-TolSCl was added directly into the reaction mixture to prevent it from freezing on the flask wall. The characteristic yellow color of p-TolSCl in the reaction solution dissipated rapidly within a few seconds indicating its depletion. After the donor was completely consumed according to TLC analysis (about 5 minutes at −78 °C), a solution of acceptor 31 (21 mg, 0.04 mmol) and TTBP (20 mg, 0.08 mmol) in CH₂Cl₂ (1 mL) was slowly added dropwise via a syringe. The reaction mixture was stirred for 1.5 h until the temperature reached 0 °C, then cooled to −78°C again. A solution of diol acceptor 4 (32 mg, 0.0125 mmol) in CH₂Cl₂ (1 mL) was added, followed by AgOTf (26 mg, 0.1 mmol) in acetonitrile (0.1 mL). After 5 minutes, p-TolSCl (6.1 µL, 0.04 mmol) was added and the reaction mixture was stirred for 1.5 h until the temperature reached 0 °C, at which point triethylamine (30 µL) was added to quench the reaction. The reaction mixture was diluted with CH₂Cl₂ (50 mL) and filtered through Celite. The Celite was washed extensively with CH₂Cl₂ until TLC showed no products in the filtrate. The filtrate was combined, concentrated and purified by flash column chromatography (hexanes : EtOAc = 2 : 3) to give compound 2 (42 mg, 64.5%). [α]_D²⁰ = −2.5 (c = 1.0 in CHCl₃). ¹H NMR (600 Hz, CDCl₃): δ = 8.20-8.12 (m, 4 H), 7.82-7.76 (m, 3 H), 7.72-6.84 (m, 103 H), 6.84-6.79 (m, 4 H), 6.78-6.74 (m, 3 H), 6.70-6.66 (m, 1 H), 6.49-6.45 (m, 1 H), 6.44-6.34 (m, 4 H), 5.56-5.40 (m, 5 H), 5.34 (s, 1 H), 5.32 (s, 1 H), 5.34-5.31 (m, 1 H), 5.24-5.19 (m, 2 H), 4.96- 4.85 (m, 6 H), 4.83-4.65 (m, 10 H), 4.63-4.41 (m, 12 H), 4.37-4.29 (m, 5 H), 4.28-4.19 (m, 8 H), 4.17-3.98 (m, 23 H), 3.94-3.83 (m, 7 H), 3.81-3.72 (m, 5 H), 3.68 (s, 2 H), 3.66-3.57 (m, 7 H), 3.56 (s, 3 H), 3.54-3.48 (m, 5 H), 3.47 (s, 3 H), 3.45-3.37 (m, 4 H), 3.33-3.15 (m, 8 H), 3.13-3.05 (m, 2 H), 2.90 (d, ³J (H, H) = 10.2 Hz, 1 H), 2.86 (d, ³J (H, H) = 10.8 Hz, 1 H), 2.77 (dd, ³J (H, H) = 5.4, 10.8 Hz, 1 H), 2.68 (dd, ³J (H, H) = 4.2, 12.6 Hz, 1 H), 2.64-2.56 (m, 2 H), 2.20 (s, 3 H), 2.19 (s, 3 H), 1.97 (s, 6 H), 1.95 (s, 3 H), 1.94 (s, 3 H), 1.91 (s, 3 H), 1.90 (s, 3 H), 1.65-1.56 (m, 2 H), 0.93 (d, ³J (H, H) = 6.6 Hz, 3 H). ¹³C NMR (150 Hz, CDCl₃): δ = 170.9, 170.6, 170.5, 170.4, 170.3, 168.68, 168.65, 168.5, 168.11, 168.05, 168.00, 164.98, 164.90, 162.3, 139.4, 139.3, 139.1, 139.04, 138.99, 138.90, 138.82, 138.6, 138.5, 138.3, 138.2, 137.9, 137.8, 137.3, 133.7, 133.6, 132.1, 132.0, 131.1, 130.3, 130.2, 130.0, 129.2, 129.1, 129.0, 128.9, 128.6, 128.5, 128.42, 128.41, 128.36, 128.33, 128.27, 128.24, 128.22, 128.17, 128.14, 128.07, 128.02, 127.90, 127.82, 127.79, 127.76, 127.66, 127.57, 127.54, 127.45, 127.34, 127.23, 127.17, 127.00, 126.97, 126.91, 126.61, 126.59, 126.52, 125.41, 123.31, 101.9, 101.5, 101.5, 101.0, 98.5, 97.7, 97.10, 97.08, 96.98, 96.96, 96.85, 95.7, 92.4, 92.1, 81.6, 81.4, 80.3, 79.3, 78.3, 78.0, 77.0, 76.2, 76.1, 75.5, 75.0, 74.9, 74.7, 74.4, 74.3, 74.1, 74.0, 73.8, 73.40, 73.35, 73.33, 73.24, 73.1, 72.8, 72.7, 72.54, 72.45, 72.1, 72.0, 70.9, 70.6, 70.3, 70.1, 69.7, 69.4, 68.8, 68.4, 68.3, 67.8, 67.6, 67.3, 66.7, 66.6, 66.1, 62.7, 62.6, 56.8, 56.2, 56.0, 53.3, 53.2, 51.6, 38.9, 38.9, 21.6, 20.9, 20.9, 20.82, 20.76, 16.7. MALDI-MS: Calcd for C₂₇₉H₂₈₀Cl₆N₆NaO₈₀, [M+Na]⁺, 5232.61 (one of the highest isotope peaks), found: 5232.24.

p-Tolyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-α-d-galacto-2-nonulopyranosonate-(2→3))-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosonyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (3)

See procedure for compound 2. The one pot reaction for synthesis of compound 2 was terminated prior to addition of acceptor 4. Compound 3 was purified from the reaction mixture by flash column chromatography (hexanes : EtOAc : CH₂Cl₂ = 4 : 3 : 3) in 82% yield. ¹H NMR (600 MHz, CDCl₃): δ = 8.15-8.12 (m, 2 H), 7.87-7.84 (m, 1 H), 7.81-7.77 (m, 1 H), 7.70-7.66 (m, 2 H), 7.63-7.59 (m, 1 H), 7.51-7.47 (m, 2 H), 7.46-7.42 (m, 2 H), 7.36-7.28 (m, 6 H), 7.28-7.24 (m, 2 H), 7.20-7.16 (m, 2 H), 6.93-6.90 (m, 2 H), 6.58 (d, ³J (H, H) = 9.6 Hz, 1 H), 5.56-5.46 (m, 3 H), 5.30 (s, 1 H), 5.21 (dd, ³J (H, H) = 1.8, 9.6 Hz, 1 H), 4.92-4.85 (m, 1 H), 4.76 (d, ³J (H, H) = 7.8 Hz, 1 H), 4.53 (dd, ³J (H, H) = 3.6, 10.2 Hz, 1 H), 4.43-4.38 (m, 2 H), 4.30 (dd, ³J (H, H) = 2.4, 12.6 Hz, 1 H), 4.22-4.06 (m, 7H), 4.05-4.00 (m, 1 H), 3.81-3.74 (m, 1 H), 3.68-3.64 (m, 1 H), 3.63 (s, 1 H), 3.61-3.57 (m, 1 H), 3.52-3.45 (m, 5 H), 2.63 (dd, ³J (H, H) = 4.2, 12.6 Hz, 1 H), 2.22 (s, 3 H), 2.20 (s, 3 H), 2.00 (s, 3 H), 1.95 (s, 3 H), 1.91 (s, 3 H), 1.62 (t, ³J (H, H) = 12.6 Hz, 1 H). ¹³C NMR (150 MHz, CDCl₃) δ = 171.0, 170.59, 170.55, 170.1, 168.7, 168.2, 168.0, 165.1, 162.3, 138.8, 138.2, 137.7, 134.24, 134.18, 133.6, 133.4, 132.1, 132.0, 130.2, 130.1, 129.7, 129.2, 128.8, 128.5, 128.41, 128.35, 127.68, 127.66, 126.6, 123.9, 123.5, 101.7, 101.0, 96.9, 92.1, 83.6, 82.1, 78.2, 73.1, 72.8, 72.3, 72.0, 71.2, 70.0, 68.7, 68.6, 67.7, 67.5, 67.3, 66.7, 62.9, 60.7, 55.4, 53.2, 51.6, 38.8, 21.7, 21.31, 21.30, 20.9, 20.8. HRMS: calcd for C₆₈H₆₉Cl₃N₂O₂₄SNa, [M+Na]⁺,1457.2924, found: 1457.2908. The Gal β1→4 GlcN linkage was confirmed by a HMBC correlation between C₁ of the galactose and H₄ of the glucosamine unit.

5-Acetamido-3,5-dideoxy-d-glycero-α-d-galacto-2-nonulopyranosylonate-(2→3)-β-d-galactopyranosyl-(1→4)-2-amino-2-deoxy-β-d-glucopyranosyl-(1→2)-α-d-mannopyranosyl-(1→6)-(5-acetamido-3,5-dideoxy-d-glycero-α-d-galacto-2-nonulopyranosylonate-(2→3)-β-d-galactopyranosyl-(1→4)-2-amino-2-deoxy-β-d-glucopyranosyl-(1→2)-α-d-mannopyranosyl-(1→3))-β-d-mannopyranosyl-(1→4)-2-amino-2-deoxy-β-d-glucopyranosyl-(1→4)-(α-l-fucopyranosyl-(1→6))-2-amino-2-deoxy-d-glucopyranoside (1)

Lithium iodide (200 mg) was added to a solution of compound 2 (80 mg) in dry pyridine (7 mL). The reaction mixture was refluxed at 120°C for 8 h under nitrogen atmosphere. The dark yellow solution was then evaporated to dryness and co-evaporated with toluene to yield a dark yellow amorphous solid which was directly used for next reaction. A solution of the above solid in ethanol (5 mL) was treated with NH₂-NH₂·H₂O (1 mL) at 85°C for 48 h. The reaction mixture was concentrated and co-evaporated with toluene then selective acetylated with Ac₂O (0.3 mL), Et₃N (0.3 mL) in methanol (3 mL) at room temperature overnight. The acetylated mixture was concentrated and passed through a short column of silica gel and eluted with CH₂Cl₂/MeOH to give compound 22. A mixture of 22, 10% Pd(OH)₂/C (30 mg) in methanol (3 mL) and water (1 mL) was stirred for 24 h at room temperature under H₂ atmosphere. The solid was filtered off and the solution was concentrated to give compound 1 (18 mg, 49%). [α]_D²⁰ = −4.2 (c = 1.0 in H₂O). ¹H NMR (600 Hz, CDCl₃): α:β mixture at the reducing end fucose δ 4.97 (d, ³J (H, H) = 2.4 Hz, 0.5 H), 4.91 (s, 1 H), 4.72 (s, 1H), 4.70-4.68 (m, 1 H), 4.57-4.54 (m, 1 H), 4.47-4.44 (m, 1 H), 4.38-4.33 (m, 4 H), 4.08 (bs, 1 H), 4.02 (bs, 1 H), 3.94-3.88 (m, 4H), 3.80-3.28 (m, 68H), 2.58 (dd, ³J (H, H) = 3.6, 12.0 Hz, 2 H), 1.88-1.82 (m, 12 H), 1.62 (t, ³J (H, H) = 12.0 Hz, 2 H), 1.06-1.02 (m, 3 H). ESI-MS: (neg. mode) Calcd for C₉₀H₁₄₈N₆O₆₆ (M-2H)²⁻: 1183.42, found: 1183.83.

Supplementary Material

Supporting info

Supporting information for this article is available on the WWW under http://www.chemeurj.org/ or from the author.